. This proposal has as its long-term objective the elucidation of the molecular mechanisms that regulate the expression of two important specialized gene systems in human erythroid cells: the globin genes that are expressed at high levels during the later stages of erythroid differentiation and the erythropoietin receptor (EPO-R) gene that is expressed at relatively low levels during the early stages of erythroid differentiation. The cis- and trans-acting factors that regulate the expression of these two behaviorly different yet erythroid specific gene systems will be compared and contrasted. The particular aspect of globin gene regulation that will be investigated is the switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA), using the transgenic mouse as a model system for hemoglobin switching. Large DNA fragments containing both fetal (gamma) and adult (beta) human globin genes have been shown by ourselves and others to be expressed in a regulated fashion (i.e., they switch during development) in transgenic mice. Whereas others have emphasized the role in this process of the locus activating region (LAR) located upstream of the globin gene, our studies will focus primarily on the role of the DNA sequences within the 40 kb KpnI DNA fragment encompassing the gamma, delta and beta globin genes. Studies will be carried out using both normal DNA fragments and corresponding DNA fragments isolated from individuals with persistent adult expression of Hb F due to different forms of nondeletion or deletion type hereditary persistence of fetal hemoglobin or delta-beta thalassemia. Studies of the human EPO-R gene will consist of the characterization of the genomic structure of the cloned chromosomal gene with particular emphasis on the structure and function of the promoter region of the gene. The general methodology to be utilized include: cloning and DNA sequence analysis of the EPO-R gene; studies of cis-acting elements by gene manipulation followed by gene retardation assays, DNase I footprinting and methylation interference techniques; studies DNase I hypersensitive sites in neighboring and remote flanking DNA for identification of possible LAR sequences; testing of functional activity of potential LAR sequences in tissue culture cells and transgenic mice. These studies should lead to a more thorough understanding of the molecular mechanisms by which regulation of globin gene expression occurs during development and perhaps indicate future approaches for the stimulation of Hb F production in individuals with hemoglobinopathies who might benefit clinically from such a process. In more general terms, the comparison of molecular mechanisms regulating EPO-R gene expression to those regulating globin gene expression should provide new insights on the process of tissue-specific gene expression during erythroid cell differentiation.